Project Summary Histoplasma capsulatum (Hc) is a thermally dimorphic fungus that is thought to be the most common cause of fungal respiratory infections in healthy humans. Hc grows in a multicellular hyphal form in the environment. Once inhaled by mammals, Hc converts to a unicellular yeast form that colonizes macrophages. Temperature is a key signal that is sufficient to trigger the switch from the soil to host form (and vice versa); in the laboratory, room temperature promotes hyphal (mold-form) growth whereas 37C promotes yeast-phase growth. The long-term goal of this research is to determine the molecular basis of how temperature regulates morphology and virulence in thermally dimorphic fungi. By elucidating how Hc cells sense and respond to host temperature, we will define critical molecular landmarks that promote changes in morphology as well as the expression of virulence traits. In previous work, we identified four transcription factors, Ryp1, 2, 3, and 4, that promote yeast-form growth in response to host temperature. The Ryp proteins are absolutely required for yeast- phase morphology as well for the vast majority of the temperature-dependent gene expression program. Over the last funding period, we uncovered evidence that Ryp1 and Ryp2 are regulated post-transcriptionally. Additionally, to identify regulatory mechanisms that antagonize the Ryp pathway at low temperature, we isolated yeast-locked mutants that inappropriately activate the Ryp pathway in the absence of the normal high temperature signal. We determined that the cell surface signaling mucin Msb2 is required for (1) inhibition of Ryp accumulation and (2) establishment of hyphal growth in response to low temperature. Additionally, transcriptional profiling of the msb2 mutant allowed us to identify compact gene regulons that are associated with either hyphal formation or yeast-phase growth, including the identification of putative virulence factors whose expression is associated with growth in the yeast form independent of temperature. Here we will (1) investigate how temperature regulates the Histoplasma pathogenic program by elucidating the molecular mechanisms that regulate the Ryp pathway; (2) elucidate how the Ryp and Msb2 pathways, which oppose each other, are able to sense temperature; and (3) utilize our temperature-defined regulatory circuits for virulence gene discovery. These experiments will result in a detailed molecular understanding of how temperature triggers critical cell fate changes that are linked to the ability of Hc to cause disease.